The objective of this project is to develop high throughput proteomic tools to enhance the study of trypanosomes - protozoan parasites that cause major public health and economic problems across the developing world. The genomic sequence of three different trypanosomes has been completed, so the next step is the characterization of all the proteins in these organisms. Affinity purification and mass spectrometry are powerful tools that are routinely used to characterize proteins, and have become virtually indispensable for proteomics research. However, both methods require appropriate sample preparation to yield quality results, and the sheer number of proteins that are encoded by eukaryotic genomes means that high throughput methods are required to rapidly and systematically analyze all possible interactions made by individual proteins. For these reasons, we are adapting and developing techniques used in the study of yeast to trypanosome research, such as: (i) cryolysis, a method for lysing frozen cells in order to preserve protein complexes as they were at the time of collection;(ii) a 96-well high throughput screen to determine optimal buffer conditions for any protein complex in a fast and facile procedure using minimal amount of cellular material;(iii) Isotopic Differentiation of Interactions as Random or Targeted (I-DIRT) - a proven method for distinguishing between specifically and nonspecifically interacting proteins using stable isotopic labeling. To develop and validate these methods in trypanosomes, we have chosen to use as a test bed a select number of component proteins (termed Nups) of nuclear pore complexes (NPCs). NPCs are the sole mediators of exchange between the nucleus and the cytoplasm;each NPC is a ~50MDa macromolecular assembly composed of 30 different Nups present in a total of ~480 copies. We chose the NPC because it represents a wide variety of protein-protein interaction types (as it is involved in nuclear transport, ribonucleoprotein complex assembly, cell cycle control and chromatin modifying complexes) and also because we previously identified and GFP-tagged 22 Nups in Trypanosoma brucei (TbNups), such that all now carry a convenient affinity handle. We will first optimize the methods using all identified TbNups. We will then focus on TbNup92, a Nup that relocates to the spindle organizer during mitosis. Successful definition of a spindle proteome will demonstrate our proteomic approach can access highly dynamic, cell cycle regulated processes. Next, we will expand these proteomic tools to explore the lamina;a meshwork of filaments within the nucleus that are intimately associated with the nuclear envelope and are involved in the regulation of nuclear structure and functions such as chromatin organization and gene transcription. Finally, we will test these methods on select targets from additional trypanosome organelles, to confirm they are broadly applicable, and ensure transferability to collaborating laboratories. With the wealth of information available from genome sequences and protein databases, we believe the methods we seek to develop will be a useful addition not only to proteomic studies in trypanosomes, but ultimately to other parasitic protists. PUBLIC HEALTH RELEVANCE: Trypanosomes cause devastating diseases in humans such as sleeping sickness. We are working to develop more efficient tools with which to study the protein interactions that are fundamental to the survival of these parasites. We believe that our methods will ultimately lead to the discovery of new drug targets to combat these disease-causing parasites.